JP2023516515A - Compositions to replace chemical surfactants - Google Patents

Abstract

The present invention provides methods and compositions for replacing chemical surfactants for use in various industrial applications. More specifically, the present invention provides for the preparation of multifunctional biosurfactant compositions having one or more precise functional properties based on the desired application.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from US Provisional Patent Application No. 62/987,529, filed March 10, 2020, the contents of which are incorporated by reference.

Surfactants are surface-active, amphiphilic molecules with potential applications in almost every industrial sector. Therefore, the market for surfactants, which now consists of thousands of different surface-active molecules, is growing rapidly. About 60% of surfactants are used as detergents and compounds for personal care products. Other applications include, for example, pharmaceuticals and supplements, oil and gas recovery, bioremediation, agriculture, cosmetics, coatings and paints, textile manufacturing, food manufacturing and processing, construction.

The properties of surfactant molecules can be measured by the hydrophilic-lipophilic balance (HLB). HLB is the size and strength balance of the hydrophilic and lipophilic portions of the surfactant molecule. A certain HLB value is required, for example, to form a stable emulsion. In water/oil and oil/water emulsions, the polar moieties of the surface active molecules are oriented towards the water and the non-polar groups are oriented towards the oil, reducing the interfacial tension between the oil and water phases. Lower.

HLB values range from 0 to about 20, with low HLB (eg, 10 or less) being oil soluble and suitable for water-in-oil emulsions, and high HLB (eg, 10 or greater) being water soluble. and is suitable for oil-in-water emulsions. Other properties such as foaming, wetting, detergency and solubilizing ability are also dependent on HLB.

Synthetic and chemical surfactants are advantageous in that they are easy to manufacture and can be tailored to perform desired functions based on their molecular structure. Thus, thousands of different surfactants have been developed, each with specific functions. While there are many options to choose from when manufacturing products in which surfactants are used, the specificity of surfactant function is such that more types and combinations are available to produce products with multiple functions. of surfactant is required. For example, surfactants useful as wetting agents are not necessarily useful as detergents, and surfactants useful as emulsifiers are not necessarily useful as anticorrosives.

The result has been the overuse and overproduction of chemical surfactants for decades. With increasing consumer and regulatory awareness, the shortcomings of chemical surfactants are beginning to surface, such as narrow activity, potential and known toxicity to humans and animals, and exposure to the environment, including aquatic environments, soils and groundwater. Persistence, contribution to climate change during manufacture and application, and incompatibility with other chemicals.

Attempts have been made to create biodegradable, low toxicity biosurfactants, but they are difficult to modify to produce products with specific physical and chemical properties. One particular group of biological surface active molecules includes those produced by microorganisms or biosurfactants. Biosurfactants are a structurally diverse group of surface-active substances consisting of two parts, a polar (hydrophilic) portion and a non-polar (hydrophobic) group.

Due to their amphipathic structure, biosurfactants can, for example, increase the surface area of hydrophobic water-insoluble substances, increase the water bioavailability of such substances, and alter the properties of bacterial cell surfaces. can. Biosurfactants can also reduce the interfacial tension between water and oil, thus reducing the hydrostatic pressure required to displace trapped liquid to counteract capillary effects. Biosurfactants accumulate at interfaces, thereby reducing interfacial tension and forming aggregated micellar structures in solution. Formation of micelles, for example, provides a physical mechanism for moving oil in a moving aqueous phase. Due to the ability of biosurfactants to form pores and destabilize biological membranes, they may also be used, for example, as antibacterial, antifungal and hemolytic agents to control the growth of pests and/or microorganisms. can.

Typically, the hydrophilic groups of biosurfactants are sugars (eg, mono-, di-, or polysaccharides) or peptides, while the hydrophobic groups are typically fatty acids. Thus, for example, sugar type, sugar number, peptide size, which amino acids are present in the peptide, fatty acid length, fatty acid saturation, additional acetylation, additional functional groups, esterification, molecular There are a myriad of potential variations of biosurfactant molecules based on their polarity and charge.

These mutations are, for example, glycolipids (e.g. sophorolipids, rhamnolipids, cellobiose lipids, mannosylerythritol lipids and trehalose lipids), lipopeptides (e.g. surfactins, iturins, phengycins, anthrofactins and lichenicins), flavolipids , phospholipids (eg, cardiolipin), fatty acid ester compounds, and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes. Each type of biosurfactant within each class can further include subtypes with more modified structures.

As with chemical surfactants, each biosurfactant molecule has its own HLB value depending on its structure, but the production of chemical surfactants resulting in a single molecule with a single HLB value or range. Unlike biosurfactant production, one cycle of biosurfactant production typically results in a mixture of biosurfactant molecules (eg, subtypes and isomers thereof), each of which has its own HLB. Thus, biosurfactant mixtures harvested from a single microbial culture typically have varying and ambiguous HLB values due to the variability of the biological processes involved in producing them.

Surfactants are an important aspect of industrial productivity around the world. Due to increasing challenges to the surfactant industry, including, for example, increasing awareness of toxicity and pollution caused by certain surfactants, environmental and health regulations, and social trends toward the desire for "environmentally friendly" products. There is a need for improved approaches for making and using surfactants.

The present invention provides methods and compositions for replacing chemical surfactants for use in various industrial applications. More specifically, the present invention provides for the preparation of multifunctional biosurfactant compositions having one or more precise functional properties based on the desired application. Advantages include, in preferred embodiments, the compositions are non-toxic, biodegradable, and environmentally friendly in manufacture and use.

In certain embodiments, customizable biosurfactant compositions are provided that comprise one or more biosurfactant molecules, wherein the identity, ratio, and/or molecular structure of the one or more biosurfactants is the desired composition of the composition. It is predetermined to achieve specific functional properties of the composition based on the application.

In certain embodiments, environmentally friendly surfactant compositions are provided that have one or more desired functional properties, the compositions comprising one or more biosurfactant molecules, wherein the one or more biosurfactant molecules Identity, ratios and structures are selected based on their contribution to the desired functional property.

In some embodiments, functional properties are measured by, for example, hydrophilic-lipophilic balance (HLB), critical micelle concentration (CMC), and/or Kauri-butanol value (KB).

In certain embodiments, the composition is selected from, for example, glycolipids, lipopeptides, flavolipids, phospholipids, fatty acid ester compounds, lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes. It comprises one or more biosurfactant molecules belonging to a class.

In some embodiments, the composition comprises multiple biosurfactant molecules belonging to the same biosurfactant class. In some embodiments, the composition comprises biosurfactant molecules belonging to two or more of these biosurfactant classes.

In some embodiments, the composition comprises glycolipids such as sophorolipids, rhamnolipids, trehalose lipids, cellobiose lipids and/or mannosylerythritol lipids. In some embodiments, the composition comprises lipopeptides such as, for example, surfactants, fengycin, anthrofactin, lichenicin, iturin and/or viscosine.

An advantage is that including multiple biosurfactant molecules in a composition in a certain predetermined ratio creates a composition with a wider range of either hydrophilicity or hydrophobicity. Additionally, the compositions are useful for multiple functions simultaneously, for example, functions requiring different HLB values or HLB ranges. That is, one biological product containing one or more biosurfactant molecules can replace a wide range of chemical products in an environmentally friendly manner (see Figure 1).

In additional and/or modified embodiments, compositions can be tailored to have specific, and sometimes very precise, HLB values based on the identities and ratios of biosurfactant molecules within the composition. can.

In certain embodiments, the composition contains chemical interfaces such as, for example, alkylbenzene sulfonates, linear alkylbenzene sulfonates, alcohol ethoxylates, diethanolamines, triethanolamines, alkylammonium chlorides, alkylglucosides, and others described herein. It can be used to replace the composition containing the active agent.

In a preferred embodiment, the present invention provides a method for making "environmentally friendly" surfactant compositions with one or more desired functional properties, the method comprising biochemicals with specific functional properties. Identifying the surfactant molecule and producing the biosurfactant molecule by culturing biosurfactant-producing microorganisms under conditions favorable for the production of the biosurfactant.

In certain embodiments, the method further comprises combining the biosurfactant molecule with one or more additional biosurfactant molecules, the identity, proportions and/or molecular structure of which are based on the desired use of the composition. It is determined. Thus, compositions are produced having one or more desired functional properties including, for example, surface/interfacial tension reduction, viscosity reduction, emulsification, demulsification, solubility, detergency, and/or antimicrobial activity.

In some embodiments, the method comprises modifying the structure of the biosurfactant molecule prior to use in the composition.

In some embodiments, the identity, proportions, and/or molecular structure of biosurfactant molecules in the eco-friendly surfactant composition is based on, for example, the HLB, CMC, and/or KB of individual molecules. It is determined. In some embodiments, biosurfactant molecule identities, proportions, and/or molecular structures are determined based on the theoretical or actual desired HLB, CMC, and/or KB values for the overall composition.

In a preferred embodiment, the environmentally friendly surfactant composition can be utilized in place of chemical surfactants in products that typically contain chemical surfactants, where one or more biosurfactants are , are selected to have the same or similar functional properties as chemical surfactants.

Accordingly, in some embodiments, the method comprises selecting a known composition comprising one or more chemical surfactants, and optionally one or more additional ingredients, and 2, making known compositions environmentally friendly by using the environmentally friendly surfactant compositions of the present invention. The environmentally friendly surfactant composition can be mixed with any additional ingredients, if present.

In some embodiments, the methods and compositions of the present invention perform better than methods and compositions that utilize competing chemical surfactants. For example, in some embodiments, the structure and/or size of biosurfactants utilized in accordance with the present invention allow for improved surface tension reduction and/or interfacial tension reduction over that achieved by chemical surfactants. to Advantageously, in certain embodiments, the biosurfactant molecules according to the invention required to achieve the desired reduction in surface tension and/or interfacial tension are required over competing chemical surfactants. It should be a lower dose than

Advantageously, the methods and compositions of the present invention completely reduce and/or replace the need for chemical surfactants, thereby reducing the costs and environmental impacts typically incurred by the manufacture and use of surfactants. is to reduce the impact of

FIG. 1 shows the HLB values of specific chemical surfactants (top) and SLP molecules (bottom). A single SLP composition (indicated by double-headed arrows with black asterisks) produced according to embodiments of the method can be substituted for multiple individual chemical surfactants. FIG. 2 shows how modification of the SLP molecule can modulate the HLB value of the molecule. Figure 3 shows how modification of a lipopeptide molecule can modulate the HLB value of the molecule. FIG. 4 shows a chart of the application of surfactant molecules and the corresponding HLB values required for the application. The chart also indicates whether more LSL or ASL is required in the composition to achieve such HLB ranges. FIG. 5 shows how modification of the RLP molecule can modulate the HLB value of the molecule. FIG. 6 shows a list of possible variations of rhamnolipid molecules with different numbers of sugar moieties and/or fatty acids, different fatty acid lengths, and different degrees of saturation in the fatty acids. Each of these 58 species has different properties, including HLB. FIG. 7 shows how deformation of a MEL molecule can modulate the HLB value of the molecule.

The present invention provides materials and methods for making "environmentally friendly" surfactant compositions that can be used in the oil and gas industry, agriculture, cosmetics, healthcare and environmental remediation, as well as various other applications. offer. Specifically, the present invention provides materials and methods for the production of universally applicable biosurfactant-derived compositions comprising one or more biosurfactant molecules, wherein the composition comprises It can be modified to exhibit one or more precise functional properties based on the type and proportion of biosurfactant molecules therein.

Advantageously, the eco-friendly surfactant compositions produced according to the methods of the present invention can exhibit specific functional properties, e.g., desired HLB, CMC and/or KB values, or desired ranges of these values. It is possible to include biosurfactant molecules in precise predetermined ratios to achieve the desired result.

Selected Definitions As used herein, "environmentally friendly" compounds or materials are compounds or materials that are at least 95% natural, biological and/or renewable, such as plants, animals, minerals and/or microorganisms. Derived from a source, furthermore the compound or material is biodegradable. Additionally, an "environmentally friendly" compound or material has minimal toxicity to humans, with an LD50>5000 mg/kg. An "environmentally friendly" product preferably does not contain any non-vegetable ethoxylated surfactants, linear alkylbenzene sulfonates (LAS), ether sulfate surfactants or nonylphenol ethoxylates (NPE).

As used herein, a "biofilm" is a complex aggregate of microorganisms, such as bacteria, yeast, or fungi, in which cells adhere to each other and/or surfaces using an extracellular matrix. Glue. Cells in biofilms are physiologically different from planktonic cells of the same organism, which are single cells that can float or migrate in liquid media.

As used herein, "isolated" or "purified" nucleic acid molecules, polynucleotides, polypeptides, proteins, or organic compounds such as small molecules (e.g., those described below) are , substantially free of other compounds such as naturally occurring cellular material. A purified or isolated polynucleotide (either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally occurring state. A purified or isolated polypeptide does not include the amino acids or sequences that flank it in its naturally occurring state. An isolated microbial strain means that the strain has been removed from the environment in which it naturally occurs. An isolated strain may thus exist, for example, as a biologically pure culture or as a spore (or other form of strain) associated with a carrier.

In certain embodiments, the purified compound is at least 60% by weight of the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 98% by weight of the compound of interest. For example, the purified compound contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired It is a compound. Purity is measured by any suitable standard method, such as column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis.

"Metabolite" refers to any substance produced by metabolism or required to participate in a particular metabolic process. Metabolites are organic compounds that are starting materials, intermediates, or end products of metabolism. Examples of metabolites include, but are not limited to, enzymes, acids, solvents, alcohols, proteins, vitamins, minerals, trace elements, amino acids, biopolymers and biosurfactants.

As used herein, "microbial-derived composition" means a composition containing components produced as a result of the growth of a microorganism or other cell culture. Thus, microbial-derived compositions can include the microorganism itself and/or by-products of microbial growth. Microorganisms may be in vegetative, spore form, mycelial form, any other form of propagule, or mixtures thereof. Microorganisms may be plankton, biofilm form, or a mixture of both. Growth byproducts are, for example, metabolites, cell membrane components, expressed proteins, and/or other cellular components. Microorganisms may be intact or dissolved. Microorganisms may be present in the composition or may be removed from the composition. Microorganisms can be present in a composition derived from microorganisms along with the broth in which they were grown. The cells are, for example, at least 1×10 ⁴ , 1×10 ⁵ , 1×10 ⁶ , 1×10 ⁷ , 1×10 ⁸ , 1×10 ⁹ , 1×10 ¹⁰ , 1×10 cells per milliliter of composition. ¹¹ , present at concentrations of multiple CFUs greater than 1×10 ¹² .

The invention further provides "products of microbial origin", which are products that are actually applied to achieve the desired result. A microbial-derived product is simply a microbial-derived composition harvested from a microbial culture process. Alternatively, the microbial-derived product may contain additional ingredients added. These additional ingredients include, for example, stabilizers, buffers, carriers such as water or salt solutions or any other suitable carrier, added nutrients to support further microbial growth, non-nutritive growth promoters. , and/or agents that facilitate tracking of microorganisms and/or compositions in the environment to which it is applied. A microbial-derived product may also include a mixture of microbial-derived compositions. A microbial-derived product may also include one or more components of a microbial-derived composition that have been processed in some manner, such as, but not limited to, filtration, centrifugation, lysis, drying, purification, and the like. good.

Ranges provided herein are understood to be shorthand for all values within the range. For example, the range from 1 to 20 is any number, combination of numbers, or subranges from the group consisting of and, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 . It is understood to include all intervening decimal values between the aforementioned integers, such as 8, and 1.9. For subranges, "nested subranges" extending from either endpoint of the range are specifically contemplated. For example, an exemplary range of 1 to 50 nested subranges may be 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 40 in the other direction. 20, and 50-10.

As used herein, "decrease" means a negative change and "increase" means a positive change, with a negative or positive change of at least 0.001%, 0.01%, 0.01%, 0.01%, 0.01%, 0.01%, 0.01%, 0.01%, 0.01% 1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

As used herein, "surfactant" refers to a compound that lowers the surface tension (interfacial tension) between two liquids or between a liquid and a solid. Surfactants act, for example, as detergents, wetting agents, emulsifiers, foaming agents and/or dispersing agents. A "biosurfactant" is a surface-active substance produced by living cells.

The phrases "biosurfactant" and "biosurfactant molecule" refer to all forms, isotopes, orthologs, isomers, and natural and/or artificial modifications.

The transitional phrase "comprising" synonymous with "having" or "containing" is inclusive or open-ended and does not exclude additional, unquoted elements or method steps. In contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not recited in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those "that do not materially affect the basic and novel characteristics" of the claimed invention. . Use of the term "comprising" encompasses other embodiments that "consist of" or "consist essentially of" the recited component(s).

Unless specifically stated or clear from context, the term "or" as used herein is understood to be inclusive. As used herein, the terms "a," "and," and "the" are understood to be singular or plural, unless specifically stated or clear from context.

Unless specifically stated or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, e.g., within 2 standard deviations of the mean. be done. About means 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.5%, 1%, 0.5%, 0.1%, 0.5% of the stated value. 05%, or within 0.01%. All numerical values provided herein are modified by the term "about," unless otherwise clear from the context.

Recitation of a listing of a chemical group in any definition of a variable provided herein includes definitions of that variable as any one group or combination of listed groups.

The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All references cited herein are hereby incorporated by reference in their entirety.

Environmentally Friendly Surfactant Compositions and Methods of Making The present invention provides environmentally friendly surfactant compositions as an alternative to chemical surfactant compositions, methods of making and methods of use thereof. More specifically, the present invention provides for the production of universally applicable biosurfactant-derived compositions comprising one or more biosurfactant molecules, wherein the compositions are biosurfactant molecule identities , proportions and/or molecular structure to exhibit one or more functional characteristics based on the desired use. In some embodiments, functional properties are measured by, for example, hydrophilic-lipophilic balance (HLB), critical micelle concentration (CMC), and/or Kauri-butanol value (KB).

In a preferred embodiment, the present invention provides a method for producing "environmentally friendly" surfactant compositions with one or more desired functional properties, wherein the method comprises biochemical agents with specific functional properties. Identifying the surfactant molecule and producing the biosurfactant molecule by culturing biosurfactant-producing microorganisms under conditions favorable for the production of the biosurfactant.

In certain embodiments, the method further comprises combining the biosurfactant molecule with one or more additional biosurfactant molecules, the identities, ratios and/or molecular structures being determined based on the desired use of the composition. be done. Thus, compositions are produced having one or more desired functional properties including, for example, surface/interfacial tension reduction, viscosity reduction, emulsification, demulsification, solubility, detergency, and/or antimicrobial activity.

In some embodiments, the identity, proportions, and/or molecular structure of biosurfactant molecules in the eco-friendly surfactant composition is based on, for example, the HLB, CMC, and/or KB of individual molecules. It is determined. In some embodiments, biosurfactant molecule identities, proportions, and/or molecular structures are determined based on the theoretical or actual desired HLB, CMC, and/or KB values for the overall composition.

One or more biosurfactants can be produced using small to large scale culture methods. Most notably, the method is suitable for use on an industrial scale, i.e., in supplying biosurfactants in amounts that meet the demand for commercial applications, e.g., the manufacture of compositions that enhance oil recovery. Can be scaled to suit. In preferred embodiments, the biosurfactant is no more than 300 miles, 200 miles, 100 miles, or 10 miles from the centralized location, in some embodiments, the location where the environmentally friendly surfactant composition is used. Produced locally, optionally modified and mixed.

Microorganisms used to produce biosurfactants may be naturally occurring microorganisms or genetically modified microorganisms. For example, microorganisms are transformed with specific genes to exhibit specific characteristics. The microorganism may also be a mutant of the desired strain. As used herein, "mutant" means a strain, genetic variant or subtype of a reference microorganism, wherein the variant has one or more genetic alterations (e.g., point mutations, missense mutations, nonsense mutations, deletions, duplications, frameshift mutations or repeat expansions). Procedures for making variants are well known in the microbiological arts. For example, UV mutagenesis and nitrosoguanidine are widely used for this purpose.

In one embodiment, the microorganism is yeast or fungus. Yeast and fungal species suitable for use in accordance with the present invention include Aureobasidium (e.g. A. pullus ), Blakeslea , Candida (e.g. C. apicola , C. bombicola , C. nodaensis ), Cryptococcus , Debaryomyces (e.g. D. hansenii ), Entomophthora , Hanseniaspora (e.g. H. uvarum ), Hansenula , Issatchenkia , Kluyveromyces (e.g. K. phaffii ), Mortierella , Meyerozyma guilliermondii , Penicillium , Phycomyces , Pichia (e.g. P. anomala , P. guilliermondii, P. occidentalis , P. kudriavzevii ), Pleurotus spp. (e.g. P. ostreatus ), Pseudozyma (e.g. P. aphidis ), Saccharomyces (e.g. S. boulardii sequela , S. cerevisiae , S. torula) , Starmerella (e.g. S. bombicola ), Torulopsis , Trichoderma (e.g. T. reesei , T. harzianum , T. hamatum , T. viride ), Ustilago (e.g. U. maydis ), Wickerhamomyces (e.g. W. anomalus ), Williopsis (e.g. W. mrakii ), Zygosaccharomyces (eg, Z. bailii ), and others.

In certain embodiments, the microorganism is a bacterium, including Gram-positive and Gram-negative bacteria. Bacteria are, for example, Agrobacterium (e.g. A. radiobacter ), Azotobacter ( A. vinelandii , A. chroococcum ), Azospirillum (e.g. A. brasiliensis ), Bacillus (e.g. B. amyloliquefaciens , B. circulans , B. laterosporus , B. licheniformis , B. megaterium , B. mucilaginosus , B. subtilis ), Burkholderia (e.g. B. thailandensis ), Frateuria (e.g. F. aurantia ), Microbacterium (e.g. M. laevaniformans ), myxobacteria (e.g. Myxococcus xanthus , Stignatella aurantiaca , Sorangium cellulosum , Minicystis rosea ) , Paenibacillus polymyxa , Pantoea (e.g. P. agglomerans ), Pseudomonas (e.g. P. aeruginosa , P. chlororaphis subsp. Rhodospirillum (eg R. rubrum ), Sphingomonas (eg S. paucimobilis ) and/or Thiobacillus thiooxidans ( Aciothiobacillus thiooxidans ).

In certain embodiments, the additional microorganism is Bacillus spp . In one specific embodiment, the Bacillus sp. is B. subtilis strain B1, B2 or B3 (see U.S. Pat. No. 10,576,519, which is incorporated by reference in its entirety), or B. subtilis subp. It is .locus B4. In certain embodiments, the Bacillus is B. amyloliquefaciens strain NRRL B-67928 (“ B.amy ”).

Cultures of B. amyloliquefaciens " B.amy " microorganisms were obtained from the Agricultural Research Service Northern Regional Research Laboratory (NRRL), 1400 Independence Ave, S.A.; W, Washington, DC, 20250, USA. The deposit was assigned accession number NRRL B-67928 by the depository and was deposited on February 26, 2020.

The cultures of the present invention are 37 CFR 1.14 and 35 U.S.C. S. C. 122 to those determined by the Commissioner of Patents and Trademarks to be entitled, under conditions ensuring that access to the culture will be available during the pendency of this patent application. The deposit will be available as required by foreign patent law in any country where the counterpart of this application or its progeny is filed. It is to be understood, however, that the availability of the deposited materials does not constitute a license to practice the invention with patent rights granted by government action expired.

Furthermore, deposits of this culture are preserved and open to the public in accordance with the provisions of the Budapest Treaty on the Deposit of Microorganisms. i.e., a minimum of 5 years after the most recent request to provide a sample for deposit, and in any event a minimum of 30 years from the date of deposit, or the enforceable duration of any patent that may disclose the culture; Preserved with all necessary precautions so as to remain uncontaminated and viable. The depositor is obliged to replace the deposit if, due to the terms of the deposit, the depository is unable to provide samples upon request. All restrictions on the availability to the public of this culture deposit will be irrevocably lifted upon the grant of a patent disclosing it.

In one embodiment, the method comprises inoculating a fermentation reactor containing a liquid growth medium with biosurfactant-producing microorganisms to produce a culture, and culturing the culture under conditions favorable for biosurfactant production.

The microbial growth vessel used according to the invention is any fermentor or culture reactor for industrial use. In one embodiment, the vessel may have functional controls/sensors or culture parameters such as pH, oxygen, pressure, temperature, agitator shaft output, humidity, viscosity and/or microbial density and/or metabolite concentration. May be connected to functional controls/sensors to measure critical factors in the process.

In further embodiments, the container also allows for monitoring of microbial growth (eg, cell count and growth phase measurements) within the container. Alternatively, samples may be taken from the vessel for counting, purity determination, biosurfactant concentration, and/or visual oil level monitoring. For example, in one embodiment, sampling can occur every 24 hours.

The microbial inoculum according to the present method preferably comprises cells and/or propagules of the desired microorganism, which can be prepared using any known fermentation method. The inoculum can be premixed with water and/or liquid growth medium if desired.

In certain embodiments, the culture method utilizes submerged fermentation in a liquid growth medium. In one embodiment, the liquid growth medium includes a carbon source. Carbon sources include carbohydrates such as glucose, dextrose, sucrose, lactose, fructose, trehalose, mannose, mannitol, and/or maltose, acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and/or pyruvic acid. alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol, and/or glycerol; canola oil, soybean oil, rice bran oil, olive oil, corn oil, sunflower oil, sesame oil, and/or linseed oil; Fats such as oil, powdered molasses, and the like. These carbon sources may be used alone or in combination of two or more. In a preferred embodiment, a hydrophilic carbon source such as glucose and a hydrophobic carbon source such as oil or fatty acid are used.

In one embodiment, the liquid growth medium contains a nitrogen source. Nitrogen sources are, for example, yeast extract, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea, and/or ammonium chloride. These nitrogen sources can be used alone or in combination of two or more.

In one embodiment, one or more inorganic salts may also be included in the liquid growth medium. Examples of inorganic salts include potassium dihydrogen phosphate, monopotassium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium chloride, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, and manganese chloride. , zinc sulfate, lead chloride, copper sulfate, calcium chloride, calcium carbonate, calcium nitrate, magnesium sulfate, sodium phosphate, sodium chloride, and/or sodium carbonate. These inorganic salts may be used alone or in combination of two or more.

In one embodiment, microbial growth factors and micronutrients are included in the medium. This is particularly preferred when growing microorganisms that are unable to manufacture all of the vitamins they require. Mineral nutrients including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt are also included in the medium. Additionally, sources of vitamins, essential amino acids, proteins and trace elements include, for example, corn flour, peptones, yeast extract, potato extract, beef extract, soybean extract, banana peel extract, etc., or in purified form. Also included are amino acids useful, for example, in protein biosynthesis.

The culture method can further provide oxygenation to the growing culture. In one embodiment, slow movement of air is used to remove hypoxic air and introduce oxygenated air. Oxygenated air is ambient air that is replenished daily through a mechanism that includes an impeller for mechanical agitation of the liquid and an air sparger to supply air bubbles to the liquid for dissolution of oxygen into the liquid. good. In certain embodiments, dissolved oxygen (DO) levels are from about 25% to about 75%, from about 30% to about 70%, from about 35% to about 65%, from about 40% to about 60% of air saturation, or maintained at about 50%.

In some embodiments, the method for culturing may further comprise adding additional acid and/or antimicrobial agents to the liquid medium prior to and/or during the culturing process. Antimicrobial agents or antibiotics (eg streptomycin, oxytetracycline) are used to protect the culture from contamination. However, in some embodiments, the metabolites produced by the yeast culture provide sufficient antimicrobial efficacy to prevent contamination of the culture.

In one embodiment, the components of the liquid culture medium are optionally sterilized prior to inoculation. In one embodiment, sterilization of the liquid growth medium can be accomplished by placing the components of the liquid growth medium in water at a temperature of about 85-100°C. In one embodiment, sterilization can be accomplished by dissolving the ingredients in 1-3% hydrogen peroxide in a 1:3 (w/v) ratio.

In one embodiment, the equipment used for culturing is sterile. Incubation devices such as reactors/vessels may be separate from the sterilization unit, eg autoclave, but may be connected. The culture device may also have a sterilization unit that is sterilized in situ before inoculation begins. Gaskets, openings, tubes and other equipment parts can be sprayed with, for example, isopropyl alcohol. Air can be sterilized by methods known in the art. For example, ambient air passes through at least one filter before being introduced into the container. In other embodiments, the medium may be pasteurized, or optionally not heated at all, and the use of pH and/or low water activity is utilized to control undesirable microbial growth. may

The pH of the culture should be suitable for the microorganism of interest and is varied as desired to produce specific biosurfactant molecules in the culture. Buffers and pH adjusting agents such as carbonates and phosphates can be used to stabilize the pH around the preferred values.

In some embodiments, the pH is from about 2.0 to about 7.0. In some embodiments, the pH is about 2.5 to about 5.5, about 3.0 to about 4.5, or about 3.5 to about 4.0. In one embodiment, culturing can be performed continuously at constant pH. In another embodiment, the culture may be exposed to varying pH values.

In one embodiment, the culturing method is carried out at about 5°C to about 100°C, about 15°C to about 60°C, about 20°C to about 45°C, about 22°C to about 30°C, or about 24°C to about 28°C. In one embodiment, culturing can be performed continuously at constant temperature. In another embodiment, the culture may be exposed to varying temperatures.

According to the method, the microorganism is allowed to remain in the fermentation system for a period of time sufficient to achieve a desired effect, e.g., production of a desired amount of cellular biomass, or a desired amount of one or more microbial growth byproducts. is incubated with Microbial growth byproducts produced by the microorganism are retained in the microorganism and/or secreted into the growth medium. The biomass content is, for example, 5 g/l to 180 g/l or more, or 10 g/l to 150 g/l.

In certain embodiments, fermentation of the yeast culture is conducted for about 48 to 150 hours, or about 72 to 150 hours, or about 96 to about 125 hours, or about 110 to about 120 hours.

After the fermentation cycle is complete, the method includes, in some embodiments, extracting, concentrating and/or purifying the biosurfactant molecules.

In certain embodiments, the methods of the present invention are performed with minimal to zero waste, thereby reducing fermentation waste discharged into sewage and wastewater systems and/or disposed of in landfills. decrease in volume.

Cellular biomass collected from the culture after biosurfactant extraction is typically inactivated and discarded. However, the method is not limited to collecting cellular biomass and using it in a live or inactive form, including but not limited to soil conditioners, livestock feed supplements, oil well treatments, and /or use for various purposes including skin care products. Cellular biomass can be used directly or mixed with specific additives for the intended application.

In some embodiments, water or other non-toxic liquids used to extract and/or purify biosurfactants may contain residual biosurfactants, nutrients and/or cellular material. Thus, in certain embodiments, the liquid is used as a soil or foliar treatment for plants, as a safe nutrition and/or hydration agent for humans and animals, as a cleaning composition, and/or as a fermentation waste. Used in irrigation drip lines or sprinklers for a myriad of other uses for reducing matter.

In some embodiments, the method comprises modifying the structure of the biosurfactant molecule prior to adding it to the composition.

In some embodiments, the fermentation parameters are adjusted to modify and/or produce one or more specific biosurfactant molecules in the culture and/or to produce a specific ratio of multiple biosurfactant molecules. becomes. These parameters include, for example, using specific strains of microorganisms, adjusting growth medium composition, co-cultivating microorganisms with antagonistic and/or influencing microorganisms, adding inhibitors and/or stimulators to nutrient media. This includes adding compounds, adjusting fermentation temperature, pH and/or aeration, and others.

In some embodiments, the biosurfactant molecule obtained from the fermentation cycle is modified post-fermentation by, for example, esterification, polymerization, addition of amino acids, addition of metals, and alteration of fatty acid chain length.

An advantage is that including multiple biosurfactant molecules in a composition in certain predetermined ratios creates a composition with a wider range of either hydrophilicity or hydrophobicity. Further, the compositions are useful for multiple functions simultaneously, eg, functions requiring different HLB values or HLB ranges. In other words, one biological product containing one or more biosurfactant molecules can be replaced in an environmentally friendly manner by a wide range of chemical products (see Figure 1).

In additional and/or alternative embodiments, the composition is tailored to have a specific, and in some cases highly accurate, HLB value based on the identity and ratio of the biosurfactant molecules within the composition. can be done.

In certain embodiments, the composition is selected from, for example, glycolipids, lipopeptides, flavolipids, phospholipids, fatty acid ester compounds, lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes. It comprises one or more biosurfactant molecules belonging to a class.

In some embodiments, the composition comprises multiple biosurfactant molecules belonging to the same biosurfactant class. In some embodiments, the composition comprises biosurfactant molecules belonging to two or more of these biosurfactant classes.

In some embodiments, the composition comprises glycolipids such as, for example, sophorolipids, rhamnolipids, trehalose lipids, cellobiose lipids and/or mannosylerythritol lipids.

In certain embodiments, the composition comprises 0% to 100%, 5% to 95%, 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 30% to 70%, 35% to 65%, 40% to 60%, 45% to 55%, or 50% by weight of sophorolipid molecules . "Sophorolipids" or "sophorolipid molecules" are e.g. acidic (linear) (ASL) and lactonic (LSL) sophorolipids, and e.g. monoacetylated, diacetylated, esterified sophorolipids , sophorolipids with varying hydrophobic chain lengths, sophorolipid-metal complexes, sophorolipids with attached fatty acid-amino acid complexes, and others described herein. can.

In certain implementations, the composition comprises 0%-100%, 5%-95%, 10%-90%, 15%-85%, 20%-80%, 25%-75% by weight rhamnolipid molecules. , 30%-70%, 35%-65%, 40%-60%, 45%-55%, or 50%. A "rhamnolipid" or "rhamnolipid molecule" includes, for example, monorhamnolipids and dirhamnolipids, all possible derivatives thereof, and other forms as described herein.

In certain implementations, the composition is 0% to 100%, 5% to 95%, 10% to 90%, 15% to 85%, 20% to 80%, 25% to 75%, 30% to It can contain 70%, 35%-65%, 40%-60%, 45%-55%, or 50% mannosylerythritol lipid molecules. A "mannosylerythritol lipid" or "mannosylerythritol lipid molecule" is, for example, triacylated, diacylated, monoacylated, triacetylated, diacetylated, monoacetylated and non-acetylated MEL, and stereoisomers and/or or may contain constitutional isomers. In certain embodiments, MEL is MEL A (diacetylated), MEL B (monoacetylated at C4), MEL C (monoacetylated at C6), MEL D (non-acetylated), triacetylated MEL A, It is characterized as a group of triacetylated MEL B/C, as well as other forms described herein.

In some embodiments, the composition comprises, by weight, 0%-100%, 5%-95%, 10%-90%, 15%-85%, 20%-80%, 25%-75%, 30% % to 70%, 35% to 65%, 40% to 60%, 45% to 55%, or 50% of lipopeptides such as surfactin, fengycin, arthrofactin, lichenicin, iturin and/or viscosine Contains peptides.

In some embodiments, two or more purified biosurfactant molecules are mixed together. In some embodiments, two or more unpurified or crude biosurfactants are mixed together. Crude forms can include, for example, residual nutrient media, microbial cells, and/or other microbial metabolites produced during fermentation. In some embodiments, a purified biosurfactant molecule is mixed with a crude biosurfactant.

In preferred embodiments, the environmentally friendly surfactant composition can be utilized in place of chemical surfactants in products typically containing chemical surfactants, wherein one or more biosurfactants are It is selected to have the same or similar functional properties as the active agent.

Thus, in some embodiments, the method selects a known composition comprising one or more chemical surfactants, and optionally one or more additional ingredients, to substitute for the chemical surfactant. to produce environmentally friendly versions of known compositions by using the environmentally friendly surfactant compositions of the present invention. The environmentally friendly surfactant composition, if present, can be mixed with one or more optional additional ingredients.

In certain embodiments, the composition can be used to replace compositions containing chemical surfactants. Typical chemical or synthetic surfactants (meaning non-biological surfactants) contain hydrophobic groups, which usually consist of long hydrocarbon chains (C8-C18) that may be branched or unbranched. while the hydrophilic groups are formed by moieties such as carboxylates, sulfates, sulfonates (anionic), alcohols, polyoxyethylenated chains (nonionic) and quaternary ammonium salts (cationic). be.

Non-biological surfactants that can be replaced in surfactant compositions utilizing the methods and compositions of the present invention include, but are not limited to, anionic surfactants, ammonium lauryl sulfate (SDS, sodium dodecyl sulfate), alkyl ether sulfates, sodium laureth sulfate (also known as sodium lauryl ether sulfate (SLES)), sodium myreth sulfate, docusate, sodium dioctyl sulfosuccinate, perfluorooctane sulfonate (PFOS), perfluoro Butane Sulfonate, Linear Alkyl Benzene Sulfonate (LAB), Alkyl-Aryl Ether Phosphate, Alkyl Ether Phosphate, Carboxylate, Alkyl Carboxylate (Soap), Sodium Stearate, Sodium Lauroyl Sarcosinate, Carboxylate Derived Fluorosurfactant, Perfluoro nonanoates, perfluorooctanoates, cationic surfactants, pH-dependent primary, secondary, or tertiary amines, octenidine dihydrochloride, permanently charged quaternary ammonium cations, alkyltrimethylammonium salts , cetyltrimethylammonium bromide (CATB) (aka hexadecyltrimethylammonium bromide), cetyltrimethylammonium chloride (CTAC), cetylpyridinium chloride chloride (CPC), benzalkonium chloride (BAC), benzonium chloride (BZT), 5-bromo -5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, cetrimonium chloride, dioctadecyldimethylammonium bromide (DODAB), zwitterionic (amphoteric) surfactant, sartein CHAPS (3-[(3-chola midopropyl)dimethylammonio]-1-propanesulfonate), cocamidopropyl hydroxysultaine, betaine, cocamidopropyl betaine, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, nonionic surfactant, ethoxylate , long chain alcohols, fatty acids, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, oleyl alcohol, polyoxyethylene glycol alkyl ethers (Brij), CH ₃ —(CH ₂ )10-16-(O—C ₂ H ₄ )1 -25-OH (octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether), polyoxypropylene glycol alkyl ether, CH ₃ —(CH ₂ )10-16-(O—C ₃ H ₆ )1-25- OH, glucoside alkyl ether, CH ₃ —(CH ₂ )10-16-(O-glucoside) 1-3-OH (decylglucoside, laurylglucoside, octylglucoside), polyoxyethylene glycol octylphenol ether: C ₈ H ₁₇ — (C ₆ H ₄ )-(O—C ₂ H ₄ )1-25-OH (Triton X-100), polyoxyethylene glycol alkylphenol ether: C ₉ H ₁₉ -(C ₆ H ₄ )-(O—C ₂ H ₄ ) 1-25-OH (nonoxynol-9), glycerol alkyl ester (glyceryl laurate), polyoxyethylene glycol sorbitan alkyl ester (polysorbate), sorbitan alkyl ester (span), cocamide MEA, cocamide DEA, dodecyl dimethyl Amine oxides, copolymers of polyethylene glycol and polypropylene glycol (poloxamers), and polyethoxylated tallowamine (POEA).

Anionic surfactants have anionic functional groups such as sulfate, sulfonate, phosphate, and carboxylate in their head. Major alkyl sulfates include ammonium lauryl sulfate, sodium lauryl sulfate (SDS, also called sodium dodecyl sulfate), and the related sodium alkyl ether sulfate, also known as sodium lauryl ether sulfate (SLES), and sodium myreth sulfate. Carboxylates are the most common surfactants and include alkyl carboxylates (soaps) such as sodium stearate.

Surfactants with cationic head groups include pH-dependent primary, secondary, or tertiary amines, octenidine dihydrochloride, permanently charged quaternary ammonium cations such as alkyltrimethylammonium salts, Cetyltrimethylammonium bromide (CTAB) also known as hexadecyltrimethylammonium bromide, cetyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), 5-bromo-5- Nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide and dioctadecyldimethylammonium bromide (DODAB).

Zwitterionic (amphoteric) surfactants have both cationic and anionic centers attached to the same molecule. The cationic moieties are based on primary, secondary, or tertiary amines or quaternary ammonium cations. The anionic portion is variable and includes sulfonates. Zwitterionic surfactants generally have a phosphate anion with an amine or ammonium, such as those found in phospholipids phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin.

Surfactants with an uncharged hydrophilic portion, such as ethoxylates, are nonionic. Many long chain alcohols exhibit some surfactant properties.

Compositions In certain embodiments, multifunctional biosurfactant-derived compositions comprising one or more biosurfactant molecules are provided. In certain embodiments, compositions can be customized for specific purposes by adjusting the identity, ratio and/or structure of the biosurfactant molecules.

In some embodiments, the identity, proportions and/or structure of the biosurfactant molecules are adjusted to achieve the desired hydrophilic-lipophilic balance (HLB). In certain embodiments, each of the individual biosurfactant molecules in the composition may be isolated to the environment, e.g., so that the composition can be used for high HLB-requiring functions and low HLB-requiring functions. acts on For example, compositions capable of oil-in-water emulsification (HLB13-18) and water-in-oil emulsification (HLB3-6) can be produced. The strength of O/W emulsification relative to the strength of W/O emulsification of exemplary compositions depends on the ratio of high HLB molecules to low HLB molecules.

In certain further and/or alternative embodiments, the biosurfactant-derived composition as a whole is characterized by a particular and, in some instances, an exact HLB value, the particular HLB value being can be tailored by adjusting the ratio of biosurfactant molecules in the

In some embodiments, the identity, proportions and/or structure of the biosurfactant molecules are adjusted to achieve a desired critical micelle concentration (CMC) value. The CMC is the concentration of surface-active molecule or composition at which micellar aggregates are formed and any additional surfactant added forms micelles. Before reaching the CMC, the surface tension decreases as the surfactant concentration increases. After reaching the CMC, the surface tension is relatively constant.

In some embodiments, the identity, proportions and/or structure of the biosurfactant molecule are adjusted to achieve the desired Kauributanol (KB) value. KB is used to represent the solvent strength of a substance as well as its detergency.

Additional ingredients can be added to the composition as needed for a particular application. Additives include, for example, buffers, carriers, other microbial-derived compositions manufactured at the same or different facilities, viscosity modifiers, preservatives, nutrients for microbial growth, nutrients for plant growth, solvents, Pharmaceuticals, nutraceuticals, tracers, pesticides, herbicides, animal feeds, disinfectants, builders, co-surfactants, fragrances, food ingredients and other ingredients specific to the intended use.

The invention further provides, for example, improved bioremediation, mining, oil and gas production, waste disposal and treatment, promotion of human health, promotion of livestock and other animal health, preservatives and/or emulsifiers. Use of these products in many situations, including as food additives, cosmetic additives, and plant health and productivity enhancement.

In some embodiments, the methods and compositions of the present invention perform better than methods and compositions that utilize competing chemical surfactants. For example, in some embodiments, the structure and/or size of biosurfactants utilized in accordance with the present invention allow for improved surface tension reduction and/or interfacial tension reduction over that achieved by chemical surfactants. do. Advantageously, in certain embodiments, the biosurfactant molecules according to the invention required to achieve the desired reduction in surface tension and/or interfacial tension are required over competing chemical surfactants. It should be a lower dose than

In certain embodiments, the size of the biosurfactant molecules and/or micelles according to the invention is less than 10 nm, preferably less than 8 nm, more preferably less than 5 nm. In certain embodiments, the size is between 0.8 nm and 1.5 nm, or between about 1.0 and 1.2 nm. Advantageously, such small size allows the biosurfactant to enter nanometer-sized spaces and pores between cells, in cell membranes, and in biofilm matrices of animals and plants, such as in oil-bearing layers in the ground. Permeation can be enhanced.

Cultivation of microbial biosurfactants according to the prior art is a complex, time-consuming and resource-consuming process, requiring multiple steps. As an advantage, the method of the present invention does not require complex equipment or high energy consumption, thus reducing the capital and labor costs of large-scale production of microorganisms and their metabolites. Additionally, surfactants made in accordance with the present invention to perform a variety of surfactant functions can be used in any application where surfactants are used, such as the oil and gas industry, the agricultural industry, and/or the cosmetic industry. only the product produced is required. Accordingly, the present invention can be used to replace and/or reduce the use of chemical surfactants in these industries.

EXAMPLES The following examples, by way of illustration, will provide a better understanding of the invention and its many advantages. The following examples are illustrative of some of the methods, applications, embodiments and variations of the present invention. It is not intended to limit the invention. Many variations and modifications can be made with respect to the invention.

Example 1 - Preparation of Sophorolipids Sophorolipids are glycolipid biosurfactants produced by, for example, various yeasts of the Starmerella clade. SLP consists of the disaccharide saphorose attached to a long-chain hydroxy fatty acid and partially acetylated 2-O-β-D β-glycosidically linked to 17-L-hydroxyoctadecanoic acid or 17-L-hydroxy-Δ9-octadecenoic acid. -glucopyranosyl-D-glucopyranose unit. Hydroxy fatty acids are generally 16 or 18 carbon atoms and contain one or more unsaturated bonds. In addition, sophorose residues are acetylated at the 6 and/or 6' positions. The fatty acid carboxyl groups may be free (acidic or linear form (general formula 1)) or internally esterified at the 4″ position (lactone form (general formula 2)). S. bombicola produces a specific enzyme called S. bombicola lactone esterase, which catalyzes the esterification of linear SLPs to produce lactone SLPs.

In a preferred embodiment, the SLPs according to the invention have general formula (1) and/or general formula (2) and have different fatty acid chain lengths (R ³ ), optionally R ¹ and/or R ² It is obtained as a collection of more than 30 types of structural homologs with acetylation or protonation at .

In general formula (1) or (2), R ⁰ may be either a hydrogen atom or a methyl group. R ¹ and R ² are each independently a hydrogen atom or an acetyl group. R ³ is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain with at least one double bond, and may have one or more substituents.

Examples of substituents include halogen atoms, hydroxyl, lower (C1-6) alkyl groups, halo-lower (C1-6) alkyl groups, hydroxy-lower (C1-6) alkyl groups, halo-lower (C1-6) alkoxy groups, and the like. Examples include, but are not limited to. R ³ typically has 11 to 20 carbon atoms, preferably 13 to 17 carbon atoms, more preferably 14 to 16 carbon atoms.

To produce SLP, the fermentation reactor is inoculated with Starmerella bombicola yeast. Keep the fermentation temperature between 23 and 28°C. After about 22-26 hours, the pH of the culture is set to about 3.0-4.0, or about 3.5 using 20% NaOH. The fermentation reactor contains a computer that monitors the pH and controls the pump used to dose the base so that the pH remains at 3.5.

After approximately 6-7 days of culture (120 hours +/- 1 hour), the batch is ready for harvest when a 7.5 ml SLP layer is visible, no oil is visible and no glucose is detected.

Modification of SLP Products During Fermentation The structure of SLP molecules produced by the methods of the invention can be modified in several ways by altering fermentation parameters. One approach is to include long chain fatty alcohols (eg, _C4 to _C26 alcohols) in the nutrient medium. The resulting SLP molecules contain hydrophobic moieties up to _C36 in length, increasing the hydrophobicity, emulsifying and detergency of the composition.

Another approach is to limit the amount of sugar and/or oil in the fermentation medium. For example, in some embodiments, the amount of glucose is limited to about 25 g/L to about 75 g/L and/or the amount of canola oil is limited to about 25 ml/L to about 75 ml/L. In certain embodiments, this increases the amount of ASL produced in the culture.

To increase the amount of hydrophobic SLP molecules (such as LSL and some ASLs), the yeast is cultured at a temperature of about 22°C to about 28°C and a pH of about 2.5-4.0. be. The pH starts at about 4.0 and decreases to stabilize at about 2.5 during the culture.

Yeast is cultivated at a pH of about 5.5 and a temperature of about 35° C. to increase the amount of ASL in the culture. Furthermore, by using the yeast Candida kuoi , a composition containing only ASL can be obtained. This is because this yeast produces only ASL.

Modification of SLP Products After Fermentation Several modifications of SLP molecules occur after the culture cycle is completed. For example, inorganic acids, alkaline substances and/or salts can be mixed with SLP to alter solubility.

Furthermore, in addition to SLPs, yeast also produce enzymes such as lipases and esterases in yeast cultures. Certain enzymes catalyze the attachment of amino acids to SLP molecules. Thus, amino acids are added to the yeast culture and selected based on the amino acid characteristics and the desired characteristics of the SLP molecule. Cationic, anionic, polar and non-polar amino acids can change the properties of the SLP molecule to cationic, anionic, polar or non-polar when bound to the SLP molecule.

Additionally, certain enzymes catalyze the esterification of SLP molecules in the presence of alcohols and fatty acids.

Upon completion of the fermentation cycle, an alcohol (eg 10% v/v) selected from methanol, ethanol, isopropyl alcohol, hexanol, or heptanol is added to the yeast culture. The liquid fermentation medium preferably already contains a fatty acid source, eg canola oil. However, if specific esterification products are desired, the addition of purified forms of fatty acids such as, for example, palmitic, stearic, oleic, linoleic, linolenic, ricinoleic, lauric, and myristic acids. of fatty acids can be added.

A yeast culture containing alcohol and fatty acids is mixed for 24 hours. After 24 hours, the mixing was stopped and the culture was fermented with added alcohol, sophorose, and fatty acid esters, such as SLP esters with methanol sophorolipid oleate formed when methanol and oleic acid were used. will include

Example 2 - Sample SLP Composition Purified LSL
LSL produced and purified using a method according to embodiments of the present invention contained 83.5% SLP (45.13% LSL and 38.36% ASL). Fatty acids (7.5%) and water (9%) made up the remainder of the product. HLB was between 1.65 and 2.99.

Despite the fact that ASL was present in the purified product, they were not treated as impurities. ASL is generally hydrophilic in nature and LSL is generally lipophilic in nature. Here, ASL showed lipophilicity. Therefore, the properties of the composition were consistent with the purer LSL, especially with respect to HLB.

Purified ASL
ASL produced and purified using a method according to embodiments of the present invention contained 92% SLP (80% ASL and 12% LSL). Fatty acids (6%), glucose (2%) and water (0.5%) made up the remainder of the product. HLB was 20 or more.

Here, ASL showed typical hydrophilicity, but small amounts of lipophilic LSL are treated as impurities that can be removed by further purification.

Example 3 - Tailoring Compositions Containing SLPs for Desired Functions The following principles are referenced when tailoring the type and/or ratio of SLP molecules in a composition (Table 1, see also Figure 2) ).

Example 4 - Analysis of SLP Compositions
Fermentation of S. bombicola was repeated 50 times to produce 50 lots of SLP with the following LSL to ASL ratios (Table 2).

A linear regression analysis was performed using the percentage of ASL in the lot. The following formula was obtained. 0.17 + (0.365x% of ASL) = HLB value

This formula can be used to predict HLB values using the percentage of ASL in SLP. Different ratios of LSL and ASL were mixed to see how the real data fit the hypothetical curve. The results support the formula as shown below (Table 3).

Example 5 - Tailoring Compositions Comprising RLPs for Desired Functions In some embodiments, compositions comprise rhamnolipids (RLPs). Rhamnolipids comprise a glycosyl headgroup (ie, rhamnose) moiety and a 3-(hydroxyalkanoyloxy)alkanoic acid (HAA) fatty acid tail, eg, 3-hydroxydecanoic acid. There are two main subtypes of rhamnolipids, mono- and di-rhamnolipids, which contain one or two rhamnose moieties, respectively. The HAA portion can vary in length and degree of branching, depending, for example, on the growth medium and environmental conditions.

式中、ｍは２、１又は０であり、ｎは１又は０であり、Ｒ¹ 及びＲ² は、互いに独立して、２～２４個、好ましくは５～１３個の炭素原子を有する同一又は異なる有機官能基、特に、置換又は非置換、分岐又は非分岐アルキル官能基であり、これらは不飽和であってもよく、アルキル官能基は、８～１２個の炭素原子を有する線状飽和アルキル官能基であるか、又はノニルもしくはデシル官能基もしくはそれらの混合物である。 A rhamnolipid according to the invention can have the following structure:

wherein m is 2, 1 or 0, n is 1 or 0, R ¹ and R ² are each independently the same having 2 to 24, preferably 5 to 13 carbon atoms or different organic functional groups, in particular substituted or unsubstituted, branched or unbranched alkyl functional groups, which may be unsaturated, the alkyl functional groups being linear saturated with 8 to 12 carbon atoms. Alkyl functional groups, or nonyl or decyl functional groups or mixtures thereof.

Salts of these compounds are also included in the present invention. In the present invention, the term "di-rhamnolipid" is understood to mean a compound of the above formula wherein n is 1 or a salt thereof; It is understood to mean a compound of a certain general formula or a salt thereof.

As shown in Figures 3-4, the structure of RLP molecules can greatly affect function.

Example 6 - Tailoring Compositions Comprising MEL for Desired Functionality -erythritol or 1-OBD-mannopyranosyl-meso-erythritol, and fatty acid and/or acetyl groups as hydrophobic moieties.

MEL subtypes can contain different carbon lengths or different numbers of acetyl and/or fatty acid groups. MEL subtypes include, for example, tri-acylated, di-acylated, mono-acylated, tri-acetylated, di-acetylated, mono-acetylated and non-acetylated MEL, as well as stereoisomers and/or or its constituent isomers. Additionally, there may be chain lengths of 1-3 esterified fatty acids, 6-12 carbons, or more.

式中、Ｒ_２及びＲ_３＝Ｃ_２－Ｃ_１８の脂肪酸、ＭＥＬ Ａ：Ｒ_４＝Ｒ_６＝アセチル基、ＭＥＬ Ｂ：Ｒ_４＝Ｈ、Ｒ_６＝アセチル基、ＭＥＬ Ｃ：Ｒ_４＝アセチル基、Ｒ_６＝Ｈ及びＭＥＬ Ｄ：Ｒ_４＝Ｒ_６＝Ｈ In certain embodiments, MEL is MEL A (diacetylated), MEL B (monoacetylated at C4), MEL C (monoacetylated at C6), MEL D (non-acetylated), triacetylated MEL A, It is characterized as a group further comprising triacetylated MEL B/C and all possible isomers of portions of these groups. A MEL according to the invention can have the following structure.

wherein R ₂ and R ₃ = C ₂ -C ₁₈ fatty acid, MEL A: R ₄ = R ₆ = acetyl group, MEL B: R ₄ = H, R ₆ = acetyl group, MEL C: R ₄ = acetyl groups, R ₆ =H and MEL D: R ₄ =R ₆ =H

As shown in FIG. 5, the structure of MEL molecules can greatly affect their function.

Example 7 - Tailoring Compositions Comprising Lipopeptides for Desired Functions In some embodiments, compositions comprise lipopeptides. Lipopeptides are oligopeptides synthesized by bacteria using large multienzyme complexes. It is often used as an antibiotic compound and exhibits broad antibacterial spectrum in addition to surfactant activity. All lipopeptides share a common cyclic structure consisting of β-amino or β-hydroxy fatty acids incorporated into the peptide portion.

Surfactin lipopeptides consist of heptapeptides containing β-hydroxy fatty acids with 13-15 carbon atoms. Phenipin lipopeptides, including privastatin, are decapeptides with β-hydroxy fatty acids. The iturin lipopeptides represented by iturin A, mycosubtilin, and basilomycin are heptapeptides with β-amino acid fatty acids.

Other lipopeptides have been identified that exhibit various useful characteristics. Some examples include, but are not limited to, Kurstakin, Anthrofactin, Viscosine, Glomosporin, Amphicin, and Syringomycin.

As shown in Figure 6, the structure of a lipopeptide molecule can greatly affect function.

In certain embodiments, the lipopeptide has one of the following general structures, wherein general structure A is iturin, general structure B is surfactin, and general structure C is fengycin. have one

Example 8 - Surfactant HLB Values Based on Intended Properties Products derived from commercial surfactants are used in food manufacturing, pharmaceuticals, cosmetics, personal care products, detergents, paints, textiles, fuels, natural and synthetic oils, Used for many other uses. In agriculture, it can be used as an insecticide and/or fertilizer. They can also be used in ore enrichment, xenobiotic remediation, and oil and gas recovery.

Surfactant selection depends on the particular intended use and is determined based on HLB values. Table 4 shows exemplary HLB values based on desired properties (see also FIG. 7).

Example 9 - Intended Use - Surfactant HLB Values Based on the Petroleum Industry , removal of contaminants and/or obstructions such as paraffin, asphaltenes and scale from equipment such as tubing, liners, tanks and pumps; prevention of corrosion of oil and gas products and conveying equipment; reduction of _H2S concentration in crude oil, reduction of viscosity of crude oil, upgrading of heavy crude oil and asphaltenes to light hydrocarbon fractions, cleaning of tanks, flow lines and pipelines, selective and non-selective blockage during water flooding It is widely used in oil and gas recovery, including enhancement of oil mobility, as well as fracturing of fluids.

Surfactant selection depends on the particular intended use and is determined based on HLB values. The following are exemplary HLB values based on desired application. Advantageously, the method of the present invention provides a surface active composition that can be tailored to perform all of the functions shown in Table 5 below for oil and gas recovery.

In certain exemplary embodiments, micelle size is another advantageous aspect for using biosurfactants in the oil and gas industry. Chemical surfactants and nanoparticle-containing fluids are often used to facilitate oil recovery from pores in formations and hydraulic fractures. The size of these compounds ranges from 15-18 nm, up to about 100 nm. For example, in certain layers containing shale, layer pore sizes are often in the low nanometer range, 13-18 nm, thus utilizing biosurfactants according to the present invention that are less than 1.5 nm in size, for example. Dosing provides a means of reaching the smallest pores and moving oils that other processes cannot.

Accordingly, methods are provided for recovering oil from oil-bearing formations having pore sizes of less than 20 nm, less than 18 nm, less than 15 nm, and/or less than 13 nm, well treatments comprising biosurfactants produced according to the present invention. A fluid is introduced into the formation, the treatment fluid contacts and displaces the oil present in the pores, and the oil is displaced in greater amounts than if chemical surfactants were used in the well treatment fluid. recovered from the layer.

Example 10 - Intended Use - Surfactant HLB Values Based on the Agricultural Industry Surfactants are widely used in the agricultural industry. Surfactant selection depends on the particular intended use and is determined based on HLB values. The following are exemplary HLB values based on desired application. Advantageously, the method of the present invention provides a surfactant composition that can be tailored to perform all of the functions shown in Table 6 below for agriculture.

In certain exemplary embodiments, micelle size is another advantageous aspect for using biosurfactants in the agricultural industry. In some instances, the small micelle size allows penetration and uptake of biosurfactants, water and solubilized nutrients into the root and vascular system of plants, reduces surface tension within plants, It allows for increased transport of nutrients and water, and increased excretion of toxins and waste products from cells. Thus, increased plant health and growth can result.

Example 11 - Intended Use - Surfactant HLB Values Based on Cosmetics and Personal Care Surfactants are widely used in the cosmetics and personal care products industry. Surfactant selection depends on the particular intended use and is determined based on HLB values. The following are exemplary HLB values based on desired application. Advantageously, the method of the present invention provides surface-active compositions that can be tailored to perform all of the functions shown in Table 7 below for cosmetics and personal care.

Example 12 - Intended Use - Surfactant HLB Values Based on Cleaning Products Surfactants are widely used in domestic, institutional and industrial (HI&I) cleaning products. Surfactant selection depends on the particular intended use and is determined based on HLB values. The following are exemplary HLB values based on desired application. Advantageously, the method of the present invention provides a surface active composition that can be tailored to perform all of the functions shown in Table 8 below for HI&I cleaning products.

Example 13 - Intended Use - Surfactant HLB Values Based on Construction Surfactants are widely used in construction. Surfactant selection depends on the particular intended use and is determined based on HLB values. The following are exemplary HLB values based on desired application. Advantageously, the method of the present invention provides surface active compositions whose construction can be tailored to perform all of the functions shown in Table 9 below.

Example 14 - Intended Use - Livestock Health Based Surfactant HLB Values Surfactants are widely used for animal health. Surfactant selection depends on the particular intended use and is determined based on HLB values. The following are exemplary HLB values based on desired application. Advantageously, the method of the present invention provides a surfactant composition that can be tailored to perform all of the functions shown in Table 10 below for the health of livestock and other pets. be.

In certain exemplary embodiments, micelle size is another advantageous aspect for using biosurfactants in animal husbandry. In some instances, small micelle size allows increased penetration and uptake of biosurfactants, water, solubilized nutrients, and drugs through intestinal epithelial cells, and increased excretion of toxins and waste products from the cells. In some instances, small micelle size is also beneficial in penetrating and disrupting biofilms on surfaces, including the gut, which may be useful in enteric methanogenic biofilms as well as other pathogenic biofilms. Useful for reducing film.

The examples and embodiments described herein are exemplary only, and in view thereof various modifications or alterations will be suggested to those skilled in the art and are intended to be included within the spirit and scope of the specification. do.

All patents, patent applications, provisional applications, and publications referred to or cited herein are hereby incorporated by reference in their entirety, including drawings and tables thereof, to the extent they do not contradict the explicit disclosure herein. be done.

Claims (16)

1. A method of producing an environmentally friendly surfactant composition having one or more desired functional properties, comprising identifying a biosurfactant molecule having a particular functional property and producing said biosurfactant by culturing a biosurfactant-producing microorganism. comprising producing a surfactant molecule;
A method, wherein said functional property of said composition and/or said biosurfactant molecule is measured by HLB value, CMC value and/or KB value. further mixing said biosurfactant molecule with one or more additional biosurfactant molecules having particular functional properties in a ratio that results in said one or more functional properties desired in said environmentally friendly surfactant composition. 2. The method of claim 1, comprising: 2. The method of claim 1, wherein the structure of the biosurfactant molecule is modified during production of the biosurfactant by altering fermentation parameters during culturing of the biosurfactant-producing microorganism. 4. The method of claim 3, wherein the structure of said biosurfactant molecule is modified after manufacture of said biosurfactant. 2. The method of claim 1, wherein said biosurfactant molecule is a glycolipid or lipopeptide. 2. The method of claim 1, wherein the biosurfactant-producing microorganism is B. acillus amyloliquefaciens NRRL B-67928. 7. The method of claim 6, wherein said B. amyloliquefaciens NRRL B-67928 produces one or more lipopeptides selected from the group consisting of surfactin, lichenicin, fengycin and iturin. An environmentally friendly surfactant composition having one or more desired functional properties, comprising one or more biosurfactant molecules, wherein the identity, proportions, and structure of said one or more biosurfactant molecules are: Environmentally friendly surfactant compositions selected based on their contribution to desired functional properties. 9. The composition of claim 8, wherein said one or more biosurfactant molecules are glycolipids selected from the group consisting of sophorolipids, rhamnolipids, trehalose lipids, and mannosylerythritol lipids. 9. The composition of claim 8, wherein said one or more biosurfactant molecules are lipopeptides selected from the group consisting of surfactin, lichenicin, fengycin and iturin. 9. The composition of claim 8, wherein said one or more biosurfactant molecules are produced by Bacillus amyloliquefaciens NRRL B-67928. 9. The composition of claim 8, wherein said one or more biosurfactant molecules are produced by Starmerella bombicola . 9. The composition of claim 8, wherein said one or more biosurfactant molecules are produced by Wickerhamomyces anomalus . 9. A composition according to claim 8, used to replace and/or reduce chemical surfactants. 9. The composition of claim 8, wherein said biosurfactant has a micelle size of less than 5 nm. A method of recovering oil from an oil-bearing formation having pore sizes of less than 20 nm, wherein a well treatment fluid comprising the composition of claim 5 is introduced into said formation, said treatment fluid having pores of less than 20 nm. A method, wherein by contacting and displacing oil present in pores, said oil is recovered from said formation in greater amounts than when a chemical surfactant is used in said well treatment fluid.

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